CN106676046B

CN106676046B - Bacillus cereus and application thereof in oxidizing inorganic sulfides

Info

Publication number: CN106676046B
Application number: CN201710091746.1A
Authority: CN
Inventors: 严小军; 庄荣玉; 娄永江; 沈青青; 何小蝶; 潜丽珍; 任雨婷
Original assignee: Ningbo University
Current assignee: Hubei Yishui Environmental Protection Technology Co.,Ltd.
Priority date: 2017-02-21
Filing date: 2017-02-21
Publication date: 2020-06-02
Anticipated expiration: 2037-02-21
Also published as: CN106676046A

Abstract

Bacillus cereus and application thereof in oxidizing inorganic sulfides, belonging to the technical field of biology and environmental protection. The invention discloses an inorganic sulfide oxidizing bacterium, which is classified and named as Bacillus cereus ZJNB-B3, is preserved in China center for type culture collection and has the preservation number as follows: CCTCC NO: m2016337. The strain has high oxidizing ability to reduced inorganic sulfide under certain conditions. The bacillus cereus disclosed by the invention can be resistant to a natural environment with a pH value within a range of 4.5-9.1, and can be applied to a natural biological desulfurization system with mild conditions, such as desulfurization and harmless treatment of malodorous hydrogen sulfide gas generated in livestock slaughtering processing and feed fish meal processing.

Description

Bacillus cereus and application thereof in oxidizing inorganic sulfides

Technical Field

The invention relates to bacillus cereus, which can be applied to biological removal of reduced inorganic sulfides in a subacid to alkaline range with the pH value of 4.5-9.1 under aerobic conditions, and belongs to the technical field of biological and environmental protection.

Background

In the production of some food processing, a large amount of hydrogen sulfide waste gas (H) with malodor harmful to human body is released₂S is a reduced inorganic sulfide), causing environmental pollution. For example, in the livestock slaughtering industry in China, the main component of the foul waste gas generated in slaughtering processing workshops is hydrogen sulfide, and the concentration can be as high as 8.0mg/m³. In the processing of the animal protein feed fish meal, fish with low freshness, rotten smelly fish and shrimp and leftovers of aquatic product processing factories are used as raw materials, and a large amount of hydrogen sulfide waste gas is released in the high-temperature cooking and drying processes.

In view of the above, there is a need for a desulfurization and deodorization method capable of rapidly removing hydrogen sulfide, applicable to various facilities, and low in cost. The current deodorization methods are mainly physical, chemical and biological. The biological deodorization method is a method for removing odor by degrading malodorous substances with microorganisms. The basic principle of treating odor by microorganisms is to utilize microorganisms to absorb malodorous substances dissolved in water into microorganisms, and degrade the malodorous substances through the metabolic activity of the microorganisms under aerobic conditions, namely, to carry out oxidative decomposition on the malodorous substances to obtain nutrients and energy and realize the proliferation process of the microorganisms. The biological desulfurization and deodorization method has the advantages of high treatment efficiency, no secondary pollution, simple required equipment, convenient management and maintenance, lower operation cost than a physical method and a chemical method and the like.

Sulfide is an important substance in nature, inorganic sulfur and its compounds are widely distributed in nature, can exist in ion or molecular forms of different forms, and can be more than 30 kinds, of which only 5 inorganic sulfides have stability in solution at room temperature and one atmosphere pressure, and are SO₄ ^2-、HSO₄ ^-、S⁰、H₂S、HS^-. Other forms of sulfur such as polysulfides, thiosulfates, and polythionates can also be found in natural environments, but they are unstable. The biological cycle process and the physical and chemical process of the sulfur are closely combined to form a natural cycle circle of the sulfur. The natural circulation of sulfur is mainly realized through the biological circulation of sulfur, and comprises (1) a process that inorganic sulfide is oxidized into elemental sulfur and sulfate under the action of aerobic sulfur bacteria, which is called a sulfur element oxidation process; (2) the process in which sulfate is converted into organic sulfur under the action of bacteria is called the assimilation reduction process of sulfur; (3) under the anaerobic environment condition, the sulfate is reduced into low-valence sulfide through the action of sulfate reducing bacteria, and the process is called dissimilatory reduction process.

The bacteria capable of oxidizing inorganic sulfide in nature are various, most of them belong to chemoautotrophic type, and they can be divided into three main categories: colorless sulfur bacteria, photosynthetic sulfur bacteria, Thiobacillus. The microorganisms participate in various processes of the thionin cycle, playing an important role. The microorganisms currently used for removing hydrogen sulfide are mainly autotrophic Thiobacillus bacteria, which utilizeThe reduced sulfide is used as an energy source, so that the incomplete oxide of the sulfur is converted into sulfuric acid and other substances, and malodorous substances such as hydrogen sulfide and the like are removed. The microbial-catalyzed thionin conversion pathway is as follows: s^2-→S⁰→SO₃ ^2-→SO₄ ^2-

However, the application of autotrophic sulfur-oxidizing bacteria to remove inorganic sulfides in reduced form has the following disadvantages: 1. sulfuric acid is generated while sulfide is oxidized, so that the activity of sulfur oxidizing bacteria is inhibited, and a large amount of acid production can cause thallus autolysis to reduce the sulfur removal rate; 2. the addition of alkali for neutralization is required, increasing the cost of the treatment.

At present, no report is found on research on chemolithotroph bacillus cereus which can oxidize reduced inorganic sulfides to generate sulfates under slightly acidic to alkaline conditions with the pH of 4.5-9.1. Therefore, it is very important to screen a heterotrophic bacterial strain which is easy to culture and can efficiently remove reduced inorganic sulfides in a natural environment.

Disclosure of Invention

The invention aims to solve the problems that in the application of sulfur oxidizing bacteria in removing inorganic sulfides in the prior art, sulfuric acid is generated while oxidizing sulfides, the activity of sulfur oxidizing bacteria is inhibited, thallus autolysis is caused, and the sulfur removal rate is reduced, and provides a strain capable of tolerating the highest S^2-The bacillus cereus with the concentration of 300mg/L and the application thereof in removing reduced inorganic sulfides have the characteristic of high-efficiency desulfurization within the pH range of 4.5-9.1.

The purpose of the invention can be achieved by adopting the following technical scheme:

a Bacillus cereus ZJNB-B3 is preserved in China center for type culture Collection in 2016, 6 and 22 days, with the preservation address of Wuhan university No. 299 in the Wuhan district, Wuhan City, Hubei province, with the preservation number: CCTCC No. m2016337, identifies sample survival by 2016, 6, 30 days.

Application of Bacillus cereus ZJNB-B3 can be used for removing reduced inorganic sulfides.

Further, by applying the Bacillus cereus ZJNB-B3, the strain can grow in the pH range of 4.50-9.10 and can oxidize reduced inorganic sulfides to generate sulfates; batch culture shows that the optimal initial pH of inorganic sulfide oxidation of the strain is 7.0, the optimal temperature is 30 ℃, and the optimal initial sulfide S is^2-The concentration is 250mg/L, and the strain can reach the calibration concentration of 244mg/L S in 48h^2-The desulfurization rate is reduced to 6.32mg/L and reaches 97.4 percent.

The invention has the advantages of

The invention provides a bacillus cereus CCTCC NO: m2016337, and its use for the oxidation-reduction of an inorganic sulfide. Bacillus cereus CCTCC NO: m2016337 is a bacterium with a strong oxidizing power for reduced inorganic sulfides. The strain belongs to heterotrophic bacteria, has simple requirements on growth nutrition conditions, is quick to grow, and can grow in a wide range of pH 4.5-9.1. The strain can oxidize reduced inorganic sulfide into sulfate radical, and can remove the reduced inorganic sulfide by oxidation in an environment with pH of 4.5-9.1 without adding alkali, so that the strain can be applied to harmless removal treatment of inorganic sulfide malodorous hydrogen sulfide gas generated by livestock slaughtering processing, feed fish meal processing and other food processing.

Drawings

FIG. 1 is a scanning electron micrograph of Bacillus cereus ZJNB-B3 strain.

FIG. 2 shows that Bacillus cereus ZJNB-B3 strain is capable of tolerating the highest sulfide S^2-Concentration, cumulative sulfuric acid radical pattern produced over 48 hours.

FIG. 3 shows the oxidation of sulfide S precursors by the Bacillus cereus strain ZJNB-B3^2-The concentration is 250mg/L, the culture temperature is 30 ℃, and the physiological metabolism change curve graph is formed under the conditions of different initial pH values of 5, 6 and 7.

FIG. 4 shows the oxidation initiation of S by the Bacillus cereus ZJNB-B3 strain^2-A change curve of physiological metabolism sulfate radical under different temperature culture of 25 ℃, 30 ℃ and 35 ℃ when the concentration is 250mg/L and the optimum initial pH value is 7.

FIG. 5 shows Bacillus cereus ZJNB-B3 strainPlant in oxidation initiation S^2-The concentration is 250mg/L, the optimum initial pH value is 7, and the optimum culture temperature is 30 ℃, the sulfate radical, the sulfide and the pH value are shown in a curve chart along with time.

Detailed Description

The invention will now be elucidated with reference to fig. 1 to 5 and specific example 1:

specific example 1 origin of mutant Strain Bacillus cereus ZJNB-B3.

250g of wet sludge precipitated at the sewage discharge position of a fish meal processing plant is taken by the applicant in 2015 and Na is continuously added₂The method of S enriches and separates to obtain a strain of bacteria capable of utilizing inorganic sulfide, the identification result is Bacillus cereus XJ-2 strain, the desulfurization rate reaches 87.1%, a research paper for separating and identifying the XJ-2 strain, namely separation and identification of a hydrogen sulfide malodorous gas removal strain processed by fish meal, is published in the environmental engineering report of 9 months 2015,9 (9): 4465-4470. As the desulfurization rate of the strain XJ-2 is not very high, the conditions that the strain XJ-2 can grow at the pH value of 5.0-8.5 are all the natural growth states of thalli under a culture liquid containing no inorganic sulfide. Strain XJ-2 contains inorganic sulfide S^2-Under the condition, the highest sulfide S tolerance can be realized^2-The physiological metabolic function of the concentration and how much concentration of sulfate is produced by oxidation is not yet known.

Under the background of the previous research, the applicant carries out mutagenesis on the bacillus cereus strain XJ-2 by using an ultraviolet mutagenesis technology in 2016, obtains mutant bacteria with the desulfurization rate as high as 97.4% by screening, is identified as bacillus cereus, is named as bacillus cereus ZJNB-B3 and is deposited in the China center for type culture collection. Subsequently, the highest sulfide S tolerated by the ZJNB-B3 strain was investigated^2-In a concentration such that S is obtained in the presence of inorganic sulfides^2-Under the condition of adding S^2-The physiological metabolism parameters of the optimal initial pH value and the optimal culture temperature for generating sulfate radicals by oxidation.

The XJ-2 strain was subjected to mutagenesis with UV light:

transferring the stored XJ-2 strain to a bacteria slant culture medium, activating and culturing at 30 deg.C for 24 hr, and culturingTransferring the activated thallus Porphyrae into sterile water triangular flask containing glass beads, shaking at 30 deg.C and 150r/min to obtain single cell suspension with cell concentration of about 10⁸One per ml. And (2) irradiating the bacterial suspension for 30-60 s under an ultraviolet lamp, taking out, culturing for 24h at constant temperature of 30 ℃, then coating the bacterial suspension on a nutrient agar culture medium solid plate, culturing for 2 days at 30 ℃, selecting grown single bacterial colonies, carrying out scribing, separation and purification to obtain a plurality of mutant strains, and screening through desulfurization rate to finally obtain a mutant strain ZJNB-B3 with high desulfurization rate reaching 97.4%.

Example 2 identification of Bacillus cereus ZJNB-B3 Strain

(1) Morphological characteristics

The gram stain of the strain is positive, bacillus exists, the cell size is 1.0-1.2 multiplied by 3.0-5.0 mu m, and the form is shown in a scanning electron microscope picture 1.

(2) Molecular biological identification

16S rDNA of bacteria is subjected to PCR amplification by adopting a 16S rDNA universal primer, DNA sequencing is carried out, The obtained sequence is compared with a known sequence disclosed in GenBank of The National Center for Biotechnology Information (NCBI) in The United states by Blast software (http:// Blast. NCBI. nlm. nih. gov), The similarity of a target strain and The known Bacillus cereus LN890259.1 reaches 100 percent, and The identified strain is named as Bacillus cereus ZB-B3 (Bacillus cereus ZJNB-B3) according to The molecular biological identification result, wherein The specific gene sequence is shown in Table 1, SEQ ID NO: 1.

example 3 oxidative physiological metabolism study of Bacillus cereus ZJNB-B3 strain on sulfide compounds of different concentrations bicyclic tube slant cultures were picked up into 100ml nutrient broth medium and shake-cultured in a shaker at 30 ℃ for 18 hours to obtain a seed solution in logarithmic phase. Inoculating 5% of the seed into a total volume of 100ml of nutrient broth with pH of 7, adding Na₂An aqueous solution of S, allowing the reaction to start with S^2-The solution was analyzed for sulfide and sulfate contents and pH changes at 100, 200, 250, 300, 400mg/L at 30 ℃ for 48 hours with shaking at 150r/min every 12 hours, and the results are shown in FIG. 2. When starting S^2-At a concentration of400mg/L, the 0h initial pH of the condition is up to 9.3, under the strong alkaline condition, the bacteria are partially killed and the growth is inhibited at the initial stage of the reaction, the pH is reduced to 7.53 after 24 hours, the thalli begin to reproduce, the pH is reduced to 7.17 after 48 hours, but the generated sulfate radical is 1.9mg/L, the desulfurization efficiency is low, therefore, the strain can tolerate S at most^2-The concentration was 300 mg/L. FIG. 2 also shows the optimal initial S of inorganic sulfides in redox state of the strain^2-The concentration is 250mg/L, the condition is to oxidize S^2-The sulfate concentration produced was maximal, reaching 60.5 mg/L.

Example 4 Bacillus cereus ZJNB-B3 Strain S^2-The optimum initial pH value and the optimum culture temperature for generating sulfate radicals by oxidation are studied on physiological metabolism.

(1) The ZJNB-B3 strain was cultured at 30 ℃ at various initial pH values and the initial S was oxidized^2-The physiological metabolism of sulfate produced by accumulation at a concentration of 250mg/L is shown in FIG. 3, and the concentration of sulfate produced by accumulation is as a result of pH7 > pH6 > pH5, so that the optimum initial pH for the strain to oxidize sulfide to produce sulfate is 7.

(2) The ZJNB-B3 strain oxidized the initial S at an initial pH of 7 at various culture temperatures^2-The physiological metabolism change of the sulfate produced by accumulation at a concentration of 250mg/L is shown in FIG. 4, the concentration of the sulfate produced by accumulation is as a result of high to low, 30 ℃ is higher than 25 ℃ and higher than 35 ℃, and the optimal culture temperature for the strain to oxidize sulfide to produce sulfate is 30 ℃.

Example 5 Bacillus cereus ZJNB-B3 Strain at initial S^2-The oxidation physiological metabolism of the sulfide is studied at the concentration of 250mg/L, the optimum initial pH value and the optimum culture temperature.

The optimum initial pH of the ZJNB-B3 strain was 7, and the optimum culture temperature was 30 ℃ and the changes in sulfate radicals and pH generated by oxidation of 250mg/L of sulfide were shown in FIG. 5. The strain can be used for separating S from liquid within 48 hours^2-The concentration is reduced to 6.32mg/L, and the desulfurization rate reaches 97.4 percent.

The bacillus cereus ZJNB-B3 can oxidize sodium sulfide into sulfate radical in a wide range of pH4.5-9.1, and can realize the oxidation removal of reduced inorganic sulfide in an environment of pH4.5-9.1 without adding alkali, so that the bacillus cereus ZJNB-B3 can be applied to the harmless removal treatment of inorganic sulfide malodorous hydrogen sulfide gas generated in livestock slaughtering processing, fish meal processing or other food processing.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and its concept within the scope of the present invention.

SEQUENCE LISTING

<110> Ningbo university

<120> bacillus cereus and application thereof in oxidizing inorganic sulfides

<130>

<160>1

<170>PatentIn version 3.3

<210>1

<211>1295

<212>DNA

<213> Bacillus cereus (Bacillus cereus)

<400>1

aacacgtggg taacctgccc ataagactgg gataactccg ggaaaccggg gctaataccg 60

gataacattt tgaactgcat ggttcgaaat tgaaaggcgg cttcggctgt cacttatgga 120

tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta 180

gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 240

aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 300

tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagtg ctagttgaat 360

aagctggcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg 420

cggtaatacg taggtggcaa gcgttatccg gaattattgg gcgtaaagcg cgcgcaggtg 480

gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg 540

agacttgagt gcagaagagg aaagtggaat tccatgtgta gcggtgaaat gcgtagagat 600

atggaggaac accagtggcg aaggcgactt tctggtctgt aactgacact gaggcgcgaa 660

agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct 720

aagtgttaga gggtttccgc cctttagtgc tgaagttaac gcattaagca ctccgcctgg 780

ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 840

gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaaa 900

accctagaga tagggcttct ccttcgggag cagagtgaca ggtggtgcat ggttgtcgtc 960

agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt 1020

gccatcatta agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg 1080

atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacggt 1140

acaaagagct gcaagaccgc gaggtggagc taatctcata aaaccgttct cagttcggat 1200

tgtaggctgc aactcgccta catgaagctg gaatcgctag taatcgcgga tcagcatgcc 1260

gcggtgaata cgttcccggg ccttgtacac accgc 1295

Claims

1. A strain of Bacillus cereus (Bacillus cereus) ZJNB-B3 which is preserved in China center for type culture Collection with the preservation number as follows: CCTCC NO: m2016337.

2. The use of a strain of Bacillus cereus (Bacillus cereus) ZJNB-B3 according to claim 1, wherein: used for biologically removing inorganic sulfides.

3. The bacillus cereus strain of claim 2Application of a bacterium (Bacillus cereus) ZJNB-B3 is characterized in that: the strain can grow in a wide range of pH 4.5-9.1, and can oxidize reduced inorganic sulfides to generate sulfates; the optimum initial pH value of inorganic sulfide oxidized by the strain is 7.0, the optimum temperature is 30 ℃, and the optimum initial sulfide S is^2-The concentration is 250mg/L, and the strain can convert S within 48h^2-The concentration is reduced to 6.32mg/L, and the desulfurization rate reaches 97.4 percent.